The damaged brain is largely incapable of functionally significant structural self-repair. This is due in part to the apparent failure of the mature brain to generate new neurons (Korr, H., Adv Anat Embryol Cell Biol 61:1–72 (1980); Sturrock, R., Adv Cell Neurobiol, vol. 3, Academic Press, New York, p. 1–33 (1982)). However, the absence of neuronal production in the adult vertebrate forebrain appears to reflect not a lack of appropriate neuronal precursors, but rather their tonic inhibition and/or lack of post-mitotic trophic and migratory support. Converging lines of evidence now support the contention that neuronal precursor cells are distributed widely throughout the ventricular subependyma of the adult vertebrate forebrain, persisting across a wide range of species groups (Goldman, S., et. al., Proc Natl Acad Sci USA 80:2390–2394 (1983), Reynolds, B., et. al., Science 255:1707–1710 (1992); Richards, L., et al., Proc Natl Acad Sci USA 89:8591–8595 (1992); Kirschenbaum, B., et al., Cerebral Cortex 4:576–589 (1994); Kirschenbaum, B., and Goldman, S., Proc Natl Acad Sci USA 92:210–214 (1995); Goldman, S., The Neuroscientist 1:338–350 (1995); Goldman, S., In: Isolation, Characterization and Utilization of CNS stem cells. F. Gage, Y. Christen, eds., Foundation IPSEN Symposia. Springer-Verland, Berlin, p. 43–65 (1997); and Gage, F., et al., Ann Rev Neurosci 18:159–192 (1995); Gage, F., et al., Proc Natl Acad Sci USA 92:11879–11883 (1995)). Most studies have found that the principal source of these precursors is the ventricular zone (Goldman, S., et. al., Proc Natl Acad Sci USA 80:2390–2394 (1983); Goldman, S., J Neurosci 10:2931–2939 (1990); Goldman, S., et al., J Neuroscience 12:2532–2541 (1992); Lois, C., et. al., Proc Natl Acad Sci USA 90:2074–2077 (1993); Morshead, C., et al., Neuron 13:1071–1082 (1994); Kirschenbaum, B., et al., Cerebral Cortex 4:576–589 (1994); Kirschenbaum, B., and Goldman, S., Proc Natl Acad Sci USA 92:210–214 (1995); Kirschenbaum, B., and Goldman, S., Soc Neurosci Abstr 317.8 (1995)), though competent neural precursors have been obtained from parenchymal sites as well (Richards, L., et al., Proc Natl Acad Sci USA 89:8591–8595 (1992); Palmer et al., 1996; Pincus, D., et al., Ann Neurology 40:550 (1996)). In general, adult progenitors respond to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) with proliferative expansion (Reynolds, B., et. al., Science 255:1707–1710 (1992); Kilpatrick, T., et. al., J Neurosci 15:3563–3661 (1995)), may be multipotential (Vescovi, A., et al., Neuron 11:951–966 (1993); Goldman, S., et al., Molec Cell Neurosci 7:29–45 (1996)), and persist throughout life (Goldman, S., et al., Molec Cell Neurosci 7:29–45 (1996)). In rodents and humans, their neuronal daughter cells can be supported by brain-derived neurotrophic factor (BDNF) (Kirschenbaum, B., and Goldman, S., Proc Natl Acad Sci USA 92:210–214 (1995)), and become fully functional in vitro (Kirschenbaum, B., et al., Cerebral Cortex 4:576–589 (1994)), like their avian counterparts (Goldman, S., and Nedergaard, M., Dev Brain Res 68:217–223 (1992); Pincus, D., et al., Ann Neurology 40:550 (1996)). In general, residual neural precursors are widely distributed geographically, but continue to generate surviving neurons only in selected regions; in most instances, they appear to become vestigial (Morshead, C., et. al., J Neurosci 12:249–256 (1992)), at least in part because of the loss of permissive signals for daughter cell migration and survival in the adult parenchymal environment.
A major impediment to both the analysis of the biology of adult neuronal precursors, and to their use in engraftment and transplantation studies, has been their relative scarcity in adult brain tissue, and their consequent low yield when harvested by enzymatic dissociation and purification techniques. As a result, attempts at either manipulating single adult-delived precursors or enriching them for therapeutic replacement have been difficult. The few reported successes at harvesting these cells from dissociates of adult brain, whether using avian (Goldman, S., et al., J Neuroscience 12:2532–2541 (1992); Goldman, et. al., Molec. Cell Neurosci. 7:29–45 (1996)), murine (Reynolds, B., et. al., Science 255:1707–1710 (1992)), or human (Kirschenbaum, B., et al., Cerebral Cortex 4:576–589 (1994)) tissue, have all reported <1% cell survival. Thus, several groups have taken the approach of raising lines derived from single isolated precursors, continuously exposed to mitogens in serum-free suspension culture (Reynolds, B., et. al., Science 255:1707–1710 (1992); Morshead, C., et al., Neuron 13:1071–1082 (1994); Palmer, T., et al., Mol Cell Neurosci 6:474–486 (1995)). As a result, however, many of the basic studies of differentiation and growth control in the neural precursor population have been based upon small numbers of founder cells, passaged greatly over prolonged periods of time at high split ratios, under constant mitogenic stimulation. The phenotypic potential, transformation state and karyotype of these cells are all uncertain; after repetitive passage, it is unclear whether such precursor lines remain biologically representative of their parental precursors, or instead become transformants with perturbed growth and lineage control.
Cells of adult organs and tissues arise from dividing progenitor cells, which themselves derive from multipotential stem cells, that both divide and are able to give rise to multiple committed progenitor cell phenotypes. Stem cells express the protein telomerase, which acts to permit continued cell division by maintaining the length of chromosomal telomeres. Telomeric shortening, which occurs when telomerase levels fall, acts as a brake upon cell division and expansion (Harley et al. Nature 345:458–460 (1990) and Allsopp et al., Proc. Natl. Acad. Sci. 89:10114–10118 (1992)). Committed progenitor cells, derived from stem cells but restricted to give rise only to defined cellular subtypes, down-regulate or lose telomerase expression. Human neural progenitors typically down-regulate telomerase activity to undetectable levels by 16 weeks of gestational age (Wright et al., Proc. Natl. Acad. Sci. 89:10114–10118 (1996) and Ulaner et al., Mol. Human Reprod. 3:769–773 (1997)), or with early passage in vitro (Ostenfeld et al., Exp Neurol 164:215–26 (2000)). In the brain, examples of phenotypically restricted progenitor cells include those for oligodendrocytes, spinal cord motor neurons, midbrain dopaminergic, and basal forebrain cholinergic neurons. These clinically-important progenitor cell types have limited capacity for mitotic expansion, at least in part because of their loss of telomerase expression (Wright et al., Proc. Natl. Acad. Sci. 89:10114–10118 (1996)). As a result, it has not hitherto been possible to generate mitotically self-renewing populations of such phenotypically-restricted lineages. Thus, whereas the therapeutic utility of embryonic stem cells has suffered from the plethora of cell types generated by multipotential stem cells, the use of phenotypically restricted progenitor cells has instead been constrained by the limited expansion of which these cells are capable.
The present invention is directed to overcoming this deficiency in the art.